The actin cytoskeleton, which plays an important role in differentiated vascular smooth muscle (VSM) cells, requires close collaboration between actin and actin-binding proteins (ABPs) under physiological and pathological conditions. Our overall aim is to determine the mechanisms by which the actin architecture of VSM cells is regulated via interactions with the ABPs. The immediate goal is to define the physiological roles of caldesmon (CaD) and cortactin and their interaction in the control of smooth muscle cytoskeleton during contraction and migration. Specifically, we propose: (1) To test the hypothesis that CaD interacts with cortactin in VSM cells where the actin cytoskeleton undergoes dynamic reorganization, and that such interaction requires specific modifications on both proteins. We will carry out pull-down assays and affinity binding in A7r5 cells and intact mouse VSM tissues under agonist stimulations, and the in situ co-localization studies by immuno-microscopy. Potential post- translational modifications (e.g., phosphorylation) of these proteins will then be examined by mass spectrometry. (2) To define the structural basis of the CaD-cortactin interaction and phosphorylation on actin regulation. We will test how the phosphorylation effect on CaD is manifested through interaction with cortactin by studying the binding between cortactin and CaD in the absence and presence of actin using kinase treated or phospho-mimetic proteins. The spatial relationship in these complexes will be assessed by distance measurements and fluorescence quenching. In addition to ERK, the effect of other kinases that are known to phosphorylated CaD and cortactin will also be examined. The interaction sites will be mapped out by affinity separation coupled with proteolysis and mass spectrometric analysis, and the structure of CaD/cortactin-bound actin filaments and the effect on the mechanical properties of the filament will be determined by 3D reconstruction and flexural rigidity measurements. (3) To determine the physiological role and of the CaD-cortactin interaction and the effects on cytoskeleton. The endogenous CaD or cortactin in the A7r5 cells or VSM tissues will first be knocked down by siRNA treatment, followed by re-expression of mutant proteins to test the regulatory effect of phosphorylation. Synthetic peptides corresponding to the interaction interface will be introduced to the siRNA treated cells as decoys. The effects resulting from these modifications will be analyzed by examining the morphology of the cytoskeleton structures, podosome biogenesis, and the migratory and contractile properties. All this information is expected to enable us to mechanistically dissect the physiological roles of CaD and cortactin in the process of the actin cytoskeleton remodeling in VSM cells, which will help us to battle cardiovascular diseases such as atherosclerosis. 420 words. PUBLIC HEALTH RELEVANCE: Millions of Americans suffer from cardiovascular diseases. A major problem is atherosclerosis and restenosis, which results from pathological movement of vascular smooth muscle cells. However, owing to the complexity of the smooth muscle cells, the molecular mechanism by which the smooth muscle cell migration is controlled remains elusive. In this project we are setting out to investigate two key proteins that are involved in the smooth muscle actin dynamics and test the hypothesis that abnormal extracellular cues can modify the interaction between these proteins and induce smooth muscle cells to migrate. Information obtained will be invaluable to battle cardiovascular diseases which remain to be number one killer in the U.S.